Extraction of actinomycin a and manufacture of its diacetates



Patented June 19, 1945 EXTRACTION OF. ACTINOMYCIN A AND MANUFACTURE OF ITS DIACETATES Max Tishler, Railway, N. J., assignor to Merck & 00., Inc., Railway, N. J., a corporation of New Jersey No Drawing. Original application October 2,

1941, Serial No. 413,324. Divided and this application November 5, 1943, Serial No. 509,152

8 Claims This application is a division of co-pending application Serial No. 413,324 filed October 2, 1941.

This invention relates to new and useful improvements in bio-chemical substances and more particularly in antimicrobial substances.

In accordance with my invention antimicrobial substances are prepared from cultures of Actinomyces antibioticus. Actinomyces antibioticus is a micro-organism which occurs inter alia in soils. Specifically, it is a fungus of defined morphology characterized by spore-bearing hyphae produced in the form of straight aerial mycelium; the sporophores are arranged in clusters, no spirals being formed; the spores are nearly spherical to somewhat elliptical.

The following method was used by me for obtaining cultures of micro-organisms capable of antagonizing various specific bacteria. In accordance with this method suspensions of cultures of the specific bacteria are mixed with an aqueous agar solution and the mixture added to a suitable soil containing the antagonizing micro-organisms. Upon one to 10 days incubation, colonies of the mycin. Thus, for instance, the actinomycin B being substantially soluble in petroleum ether may be extracted as a solution in this solvent in which the actinomycin A is substantially insoluble. Alternatively, instead of extracting the actinomycin A or actinomycin B from actinomycin, they may be obtained by direct selective solvent extraction from the original cultures.

Actinomycin A is a highly pigmented (red) product substantially soluble in ether, ethyl alcohol, water, carbon bisulphide, acetone, chloroform and substantially insoluble in petroleum ether. Its aqueous solutions are of a yellow to orangered color, depending on concentration. The actinomycin B is substantially soluble in ether, carbon bisulphide, acetone, chloroform, petroleum ether, but diificultly soluble in alcohol, and substantially insoluble in water;

The actinomycin A is highly bacteriostatic,

-grampositive bacteria being inhibited by dilutions as low as 1;100,000,000; the A compound is also efiective, though not in such degree of sensitivity against gram-negative bacteria which are inantagonists will form which may be transferred to fresh bacterial nutrient media and later isolated as pure cultures in accordance with conventional methods.

By means of this method, I have isolated an organism which I have described as Actinomyces antibioticus. The method of isolation is illustrated in Example IV.

In the practical application of my invention, a suitable culture of Actinomyces antibioticus is extracted with a suitable solvent. Suitable solvents are, for instance, ethyl ether, ethyl alcohol,

carbon bisulphide, acetone, and chloroform.

\ Ethyl ether is the preferred solvent. Upon evaporation of the solvent, a solid remains. This substance, hereinafter designated, as actinomycin possesses marked bacterlostatic and bactericidal properties against a large variety of bacteria, atcinomycetes and fungi. In most cases, grampositive bacteria are much more sensitive against the substance than gram-negative organisms. The actinomycin is completely removable by charcoal from liquid media but is only partly removed therefrom by a Seitz filter. The substance is substantially heat resistant at 100 C. for thirty minutes.

I have found that two diiferent antimicrobial substances may be obtained from the actinomycin. These substances, which will be hereinafter referred to respectively as actinomycin A" and actinomycin B may be obtained by selective extraction with suitable solvents from the activehibited by concentration of, for example, 1:5,000 to 1:100,000.' The actinomycin A also possesses bactericidal activity, though not as pronounced as its bacteriostatic characteristics. The actinomycin B is markedly bactericidal and to a lesser extent bacteriostatic.

Actinomycin A crystallizes from suitable solutions, such as acetone-benzene or acetone-ether mixtures or ethyl-acetate, as vermillion 'red platelets which melt at 250 C. with slow decomposition. The actinomycin B crystallizes substantially colorless. The A component exhibits characteristic absorption in the visible and ultraviolet regions. It shows strong absorption at 450 m and between 230 and 250 m The following examples illustrate methods of obtaining actinomycin as well as its A and B components, and it is to be understood that the same is furnished by way of illustration and not of limitation.

Example I An incubated culture of Actz'nomyces antibioticus was prepared using a medium consisting of 1% tryptone-peptone, 0.5% starch, 0.2% KzHPO4, 0.2% NaCl and 0.25% agar in distilled water, grown at a temperature of approximately 25 to 35 C., the incubation being complete after six to ten days. Fifty liters of this incubated culture are extracted approximately six times with ether, using 20 liters of ether for each extraction. The final extract is faintly pale yellow in color, whereas the previous extracts are orange. The combined ether extracts are concentrated to dryness and about 3 gms. of a reddish-brown residue is obtained. The residue is stirred with aproximately 400 cc. of petroleum ether for two to three hours, the solvent decanted and the residue treated again with approximately 400 cc.

. of petroleum ether. A pale yellow oil constituting crude actinomycin B is recovered by evaporation from the petroleum ether.

The dark petroleum ether insoluble residue is dissolved in one liter of benzene with gentle heating. Usually a small amount of black amorphous material remains undissolved and is filtered off. The benzene solution is permitted to drop through a chromatographic tower (60 x 5 cm.) packed with aluminum oxide (according to Brockman), The pigment is readily adsorbed. The column is washed with about one liter of benzene during which operation very little migration of the color bands occurs.

The column is then washed with benzene acetone solution (15:85) whereby a chromatogram develops. By continued washing, light yellow colored pigments pass out of the column. When, the main band (orange-red) reaches the lower end of the column, a solution of 30-70 acetone benzene is passed through the column. The latter solvent elutes the pigment and when the eluate is very pale in color, washing is discontinued.

The eluate is concentrated to dryness under reduced pressure, taken up in 25 cc. of hot acetone, filtered, and diluted with ether. The pigment which crystallizes as red-brick colored platelets is essentially pure but may be recrystallized if desired from hot ethyl acetate. An analysis of the product showed C=59.01; H=6.81; N=13.38.

In accordance with my invention, a variety of derivatives of actinomycin A or actinomycin B may be prepared. Thus, for instance, the following example II recites, by Way of illustration, the preparation of the di-acetate of di-hydro actinomycin A.

Example II 25 mgs. of actinomycin A are dissolved in 1 cc. acetic anhydride and to this is added 3 drops of pyridine (or 3 drops triethylamine) and 100 mg. of zinc dust. On stirring, the red mixture becomes pale yellow in color. After one hour at room temperature, the mixture is filtered from the zinc dust and the filtrate is concentrated to dryness under reduced temperature. The pale yellow residue is dissolved in chloroform and washed with water, with aqueous sodium bicarbonate, and finally with dilute hydrochloric acid. The chloroform solution is concentrated to dryness under reduced pressure and the residue is taken up in 1 cc. acetone. on adding two volumes of ether, the product separates as a pale yellow solid melting at approximately 241 C. Approximate analysis: C 58.32; H 6.36; N 12.48.

Example III 25 mg. of actinomycin A in 1 cc. of pyridine and 0.5 cc. of acetic anhydride are allowed to stand ten hours at room temperature, and then for two hours at 80 C. The red solution upon concentration to dryness under reduced pressure is dissolved in chloroform and the latter solution washed in the order mentioned with dilute hydrochloric acid and aqueous sodium bicarbonate. The chloroform solution is concentrated to dryness and the resulting product recrystallized from ethyl acetate. The same constitutes red-brick prisms melting at approximately 250 C. Approximate analysis: C 58.27; H 6.56; N 12.46.

The method of growing cultures of antagonists to specific bacteria outlined above can be demonstrated by way of the following example.

Example IV Agar is, washed in distilled water and dissolved so as to yield 1.5% concentration. Two gms. of K2HPO4 are added per liter. Ten milliliter portions of this agar are distributed in test tubes and sterilized. A washed suspension of Escherichia coli.', or some other suitable bacterium, obtained by cultivation on solid or in liquid nutrient, media is prepared and added to the washed agar which has previously been melted and placed in a water bath at 42 C. One milliliter portions of the still viable bacterial suspension are added to the agar tubes and thoroughly mixed with the agar.

A suitable soil is suspended in sterile tap water using a series of dilutions from 1:10 to 1:10,000. One milliliter portions of these dilutions are placed in sterile Petri dishes and the bacterial agar, prepared as above, is added. The plates are well shaken to distribute the soil suspension thoroughly and are' incubated at 28 or 37 C. After one to ten days incubation of the plates, colonies of the antagonists will appear being surrounded by clear zones. These colonies are transferred to fresh bacterial agar plates and are later isolated in pure culture by the use of convenient media. Actinomyces antibiotz'cus is one of the antagonists isolated by this method.

I have further discovered a method and culture medium for increasing the production of actinomycin or its components by the cultures of Actinomyces antibioticus. The culture medium in accordance with this embodiment essentially comprises 1 to 10 and preferably 5 parts by weight of starch, 1 to 20 and preferably 10 parts by weight of a suitable nitrogen containing material of an organic or an inorganic nature, a small amount of buffering agent sufficient to maintain the pH of the culture substantially between pH 5.6 and pH 8 and preferably at pH 7, 1 to 5 and preferably 2 parts by weight of a mineral salt, preferably sodium chloride, and 2 to 20, preferably 15 parts by weight agar, and 1000 parts by weight of water. The buiiering agent may be any one of the known buiiering agents and preferably K2HPO4; amounts of 1 to 3 and preferably 2 parts by weight of the buffering agent will yield the desired pI-I or pH range. As a source of nitrogen a variety of sub- 1.5% agar, 0.2% K2HPO4, 0.2% NaCl, 0.5 to 2% starch, 0.1 to 0.2% asparagine;

1.5% agar, 0.2% Kai-IP04, 0.2% NaCl, 0.5% starch,

1% p ptone;

1.5% agar, 0.2% KflIIPOL, 0.2% NaCl, 0.5% starch, 1.0% tryptone;

0.25% agar, 0.2% K2HPO4, 0.2% NaCl, 1.5% starch, 0.2% NaNOs.

Cultures are preferably incubated at 25 to 35 C., substantially maintaining the pH at the indicated point or range. Growth proceeds rapidly andis accompanied by the formation of a soluble dark-brown pigment on organic media. It becomes rapidly covered with a white mycelium having a faint yellowish-green tinge. After 6- to 10 days incubation, growth and production 01' actinomycin are usually complete.

I have found that the actinomycin in accordance with my invention possesses a selective bacteriostatic action with respect to different varieties of bacteria and that the markedly greater sensitivity of some bacteria toactinomycin than of others may be utilized for thepurpose oi isolating specific organisms from a mixed population. Actinomycin has a bacteriostatic eli'ect in relatively small concentrations on gram-positive bacteria, while it requires much higher concentrations to 'be eiiective bacteriostatically with respect to gram-negative bacteria. Thus, for instance, when adding 0.1 mg. actinomycin to 10 cc. agar and incubating thereon a substrate such as milk or sewage containing both gram-positive and gram-negative bacteria, the growth of the gram-positive bacteria will be inhibited by the actinomycin. Generally, concentrations of 0.1 to 1.0 mg. per 10 cc. agar permit the selective growth 01. various bacteria .by reason of the differentlation in sensitivity of varying amounts of the active actinomycin with respect to specific bacterial growth.

Both the actinomycin A and actinomycin B possess a relatively high molecular weight. Actinomycin A has the general emprical composition or 58.7 to 59.3% C, 6.5 to 7.5% H, 13.05 to 13.65% N, and a molecular weight in excess of 700.

Modifications may be made in carrying out the present invention without departing from the spirit and scope thereof, and I am to be limited only by the appended claim.

I claim:

1. The process that comprises passing a henzene solution oi the petroleum ether insoluble residue of crude actinomycin through a chromatographic tower packed with aluminum oxide, washing the column successively with benzene and a benzene acetone mixture predominantly acetone, eluting actinomycin with a benzene acetone mixture predominantly benzene and recovering actinomycin A from the benzene acetone eluate.

2. The process that comprises passing a benzene solution of the petroleum ether insoluble residue of crude actinomycin through a chromatographic tower packed with aluminum oxide,

washing the column successively with benzene and a 15-85 benzene acetone mixture, eluting actinomycin with a 70-30 benzene acetone mixture and recovering actinomycin A from the benzene acetone eluate.

3. The process that comprises dissolving in benzene the dark petroleum ether insoluble resdue of crude actinomycin, passing the benzene solutionthrougha chromatographic tower packed with aluminum oxide, washing the column successively with benzene and a benzene acetone mixture predominantly acetone until the dark coloration reaches the end of the column, passing through the column a benzene acetone mixture predominantly benzene to elute the pigment from the column and recovering from the benzene acetone eluate red-brick colored crystals of actinomycin A.

4. The process that comprises passing a benzene solution of the petroleum ether insoluble residue of crudeactinomycin through an adsorbing medium, preferably aluminum oxide, washing the adsorbing medium successively with benzene acetone mixture predominantly acetone, eluting actinomycin from the adsorbing medium by a benzene acetone mixture predominantly benzene and recovering from the benzene acetone eluate brick red colored crystals of actinomycin A.

5. Dihydro actinomycin A diacetate, a pale yellow material melting at about 241 C, and having the approximate analysis by weight: 58.32% C, 6.36% H, 12.48% N.

6. The process that comprises reacting actinomycin A with a mixture of acetic anhydride, pyridine and zinc dust and recovering from the reaction product a pale yellow material melting at approximately 241 C. and having the approximate analysis by weight: 58.32% C, 6.36% H, 12.48% N.

7. Actinomycin A diacetate, a brick red crystalline material melting at about 250 C. and having the approximate analysis by weight: 58.27% C, 6.56% H, 12.46% N.

8. The process that comprises reacting actinomycin A with a mixture of pyridine and acetic anhydride, predominantly pyridine, and recovering from the reaction product a brick red crystalline material melting at approximately 250 C. and having the approximate analysis by weight: 58.27% C, 6.56% H, 12.46% N.

MAX TISHLER. 

